The principal aim of this proposal involves the selective isolation of focal and nodular "preneoplastic" hepatocyte populations from the livers of carcinogen-treated rats and their subsequent classification according to their relationship to the process of hepatocarcinogenesis. In this respect, specific physical and functional alterations of the "preneoplastic" hepatocytes such as alterations in their cell size and ploidy, their deficiency in storing iron, their retention of glycogen upon fasting, and their significant expression of the "fetal" liver enzyme gamma-glutamyl transpeptidase will be exploited by means of suitable pretreatments of the animals and/or by the use of separation procedures designed to isolate and separate heterogenous cells into distinct population classes. Specifically, single cell suspensions will be prepared from whole "preneoplastic" liver and from carcinogen-induced hyperplastic nodules according to an established collagenase perfusion technique. A number of approaches will then be explored to separate the "preneoplastic" hepatocytes from other liver cell types, as well as into specific subpopulations. These will include the use of centrifugation in isopycnic gradients of modified colloidal silica (Percoll), sedimentation velocity at unit gravity, and cell affinity chromatography using anti-gamma-glutamyl transpeptidase antibody coupled to agarose. Once separated into distinct classes, the "preneoplastic" hepatocytes will be characterized by their ability to be transplanted into the livers of isologous recipient rats, by their responsiveness in vivo and in culture to hepatic promoting agents, by their altered enzymatic and proliferative activities in vivo and in culture, and by their capacity to give rise to hepatocellular carcinoma.